The Institute of Medicine has identified antibiotic resistance as one of the key microbial threats to health in the United States and has prioritized decreasing inappropriate use of antimicrobials as the primary solution to address this threat. The emergence of carbapenem resistance among Enterobacteriaceae (CRE) in the United States represents a recent and severe byproduct of excessive antimicrobial use with a high mortality rate in bacteremia. A major barrier toward decreasing use of antimicrobials is lack of sensitive and accurate rapid diagnostic tests for identifying bacterial etiologies of infection. Itis our overall goal to develop a new diagnostic technology that will facilitate antibacterial stewardship to reduce selective pressure and improve patient outcomes. We hypothesize that the Accelerate ID/AST system will enable sensitive and specific, culture-independent detection of the most commonly encountered Enterobacteriaceae direct from blood, with corresponding carbapenem resistance phenotype, in less than 3 hours. Individual live cells of bacteria, extracted directly from patient blood, and immobilized in a transparent multichannel fluidic cassette enables automated microscopy to perform single-cell analysis for identification and phenotypic susceptibility testing. In Aim 1 we will validate the system in simulated positive blood cultures. In Aim 2 we will field test performance in actual patient blood samples. In Aim 3, we will complete the Accelerate ID/AST system product development process up to the point of readiness for validation in a multicenter trial. At the conclusion of these Aims, we will have produced a sensitive and specific, culture-independent detection method for rapid species identification and susceptibility result, direct from blood, in under 3 hours. Accurate and timely detection would hone management and inform infection prevention measures to prevent spread of infection that, in turn, would prevents need for more antibiotic use-- which is needed to stop feeding the vicious cycle of antimicrobial resistance.